Field of Use
The present application relates generally to cryopreservation and generally to fast cooling of biological material for use in industries such as medicine, animal husbandry, and biomedical science.
Description of the Related Art
Cryopreservation is a process where biological materials, such as cells, tissues, or entire organs are preserved by cooling them to sub-zero temperatures. At low enough temperatures, any enzymatic or chemical activity which might cause damage to the biological material is effectively halted. Cryopreservation methods seek to reach low temperatures without causing additional damage caused by the formation of ice during freezing. Traditional cryopreservation has relied on coating the material to be frozen with a class of molecules termed cryoprotectants.
Cryofixation is a similar technique for fixation or stabilization of biological materials at ultra-freezing temperatures. This method involves ultra-rapid cooling of small biological samples to the temperature of liquid nitrogen (−196° C.) or below, stopping all motion and metabolic activity, and preserving the internal structure of cells by freezing all fluid phases solid. The ultimate objective is to freeze the specimen so rapidly (at 104 to 106 K per second) that ice crystals are unable to form, or are prevented from growing big enough to cause damage to the specimen's ultrastructure. The formation of samples containing specimens in amorphous ice is considered by some to be the holy grail of biological cryomicroscopy.
One method used to accomplish cryofixation is literally “plunging” biological material into to vial of very cold liquid refrigerant. In this technique, a small vial of ethane may be placed inside a larger liquid nitrogen reservoir. Then, a plunger is positioned over the reservoir, the plunger having an electron microscopy (EM) grid positioned at the bottom of the plunger. The EM grid comprises biological material to be frozen. The plunger has a heavy weight at the top that drives the plunger in a downward direction, forcing the EM grid into the ethane very quickly. When the EM grid enters the liquid ethane, the biological sample is frozen very rapidly, and then the grid can be moved to a storage box in liquid nitrogen for later use, for example, examination under an electron-microscope.
There are a number of drawbacks, however, to the technique described above. First and foremost, the plunger and reservoir must be precisely aligned with one another, and the length of travel by the plunger must be precisely controlled. This often results in a bulky, fixed device (e.g., non-portable) that is not capable of transferring the biological material to another location. Transferability may be desired, for example, the ability to transfer the biological material to another location along with some of the liquid refrigerant to keep the biological material as cold as possible during the transfer.